Sequence-specific isothermal and polymerase chain reaction (PCR) nucleic acid amplification techniques represent rapidly growing sectors of molecular diagnostics, offering rapid, sensitive detection of DNA samples.
Electrophoresis is a traditional method of detecting DNA products in a post-amplification step that utilizes labor intensive manual processing and instrumentation. Recent developments in isothermal amplification has provided alternative detection methods, for example, fluorescence detection of double-stranded DNA (dsDNA) with an intercalating or magnesium-sensitive fluorophore (Notomi, et al., Nucleic Acids Res., 28:E63 (2000); Tomita, et al., Nat. Protoc., 3:877-82 (2008); Goto, et al., BioTechniques, 46:167-72, (2009)); bioluminescence through pyrophosphate conversion (Gandelman, et al., PLoS One, 5:e14155 (2010); or turbidity detection of precipitated magnesium pyrophosphate (Mori et al., Biochem. Biophys. Res. Commun., 289:150-4 (2001)). However, these visual methods typically require long incubation times (>60 minutes), require specific instruments for detection, or are too subtle in change for robust detection outside of the laboratory. Advances in real time PCR equipment and chemistries have allowed monitoring many samples simultaneously during the PCR reaction. The detecting principles are typically based on either using fluorescence detection of dsDNA with an intercalating dye or using sequence-specific fluorescent probes requiring costly instruments. Alternatively, instruments have been developed for detecting hydrogen ions released during polymerase dependent amplification. Detection of these hydrogen ions has been achieved using sophisticated electronic detection and microfluidic devices, for example as demonstrated in U.S. Pat. No. 7,888,015 for use in high-throughput Next Generation Sequencing (Ion Torrent™ Sequencing, Life Technologies, Grand Island, N.Y.).
Point-of-care and field diagnostics require rapid and simple tests, ideally detecting target nucleic acid in less than 30 minutes and without sophisticated and costly equipment.